Chicken egg-white avidin and bacterial streptavidin (from Streptomyces avidinhi) are tetrameric proteins which bind biotin with similar affinity constants (Ka ˜1015 M−1), the strongest protein-ligand interaction known in nature. This affinity, together with the facile incorporation of the biotin moiety into various binders and probes, has served to promote the widely used avidin-biotin technology.
Despite extensive knowledge about the properties of avidin, little is known about its biological function. Since the growth of a number of microorganisms is inhibited by avidin, and its occurrence after tissue damage has been clearly demonstrated, it most likely has a role as a host defence factor.
Enhanced cellular uptake of avidin-coated vectors has been reported (Pardridge and Boado, FEBS Lett. 288: 30-32, 1991; Vinogradov Bioconjug. Chem. 10: 851-860, 1999). Also, polylysine (net positive charge at physiological pH) has been used to pseudotype adenovirus and shown to augment gene transfer to a variety of CAR-deficient cell types, including macrophages, smooth muscle cells, fibroblasts, endothelial cells, T cells, glioma cells, acute myeloid leukemic cells, myeloma cells and skeletal muscle cells (Wickham et al., J. Virol. 71: 8221-8229, 1997; Hidaka et al., J. Clin. Invest. 103: 579-587, 1999; Yoshida et al., Hum. Gene Ther. 9: 2503-2515, 1998; Gonzalez et al., Hum. Gene Ther. 10: 2907-2717, 1999; Gonzalez et al., Gene Ther. 6: 314-320, 1999). Furthermore, it has been shown that biotin attached to cell surface membrane proteins enables efficient entry of avidin bioconjugates into nucleated cells.
Avidin has been used for drug targeting; in vivo studies have shown that avidin preferentially accumulates into certain tissues, including tumour tissue (Yao et al., J. Nat. Cancer Inst. 90: 25-29, 1998; Rosebrough and Hartley, J. Med. 37: 1380-1384, 1996).
Gene therapy is a highly potent and rapidly developing field of research aimed at treat or prevent disease by gene transfer. Currently, the most efficient vectors are viral. However, the low efficiency of gene transfer still limits successful gene therapy and there is a need for more facile and efficient gene transfer vectors. The ideal vector should also be capable of cell-specific gene delivery in order to provide the therapeutic effect where needed, in contrast to uncontrolled gene transfer to normal tissues and organs.
Although transient expression may be desired and/or sufficient in gene therapy of cancer or cardiovascular diseases, more prolonged transgene expression is needed for treatment of inherited metabolic disorders such as severe combined immunodeficiency (SCID).
The origin of replication (orip) of the Epstein-Barr virus (EBV) supports stable replication of plasmids, and the cloned cDNAs they contain, in proliferating cells from many species. In cells expressing Epstein-Barr nuclear antigen I (EBNAI), plasmids containing oriP replicate during S phase and segregate to daughter cells efficiently. The number of plasmids per cell is stable.
Baculoviruses have long been used as biopesticides (Cory and Bishop, 1997) and as tools for efficient recombinant protein production in insect cells. They are generally regarded as safe due to the naturally high species-specificity and because they are not known to propagate in any non-invertebrate host. They are incapable of replication in mammalian cells, and have a large capacity for the insertion of foreign sequences into the genome.